PHOSPHATIDYLINOSITOL 3-KINASE-DEPENDENT ACTIVATION OF PROTEIN-KINASE C-ZETA IN BACTERIAL LIPOPOLYSACCHARIDE-TREATED HUMAN MONOCYTES

The isoform identity of activated protein kinase C (PRC) and its regul ation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange c hromatography revealed two principal peaks of myelin basic protein kin ase activity. Immunoblotting and immunoprecipitation with isoform-spec ific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-zeta. In addition to primary monocytes, activ ation of PKC-zeta in response to LPS was also observed in the human pr omonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-zeta, the kinase did not depend upon the presence of lipids, Ca2, or diacylglycerol for activity. In addition, the kinase phosphorylat es peptide a and myelin basic protein with equal efficiency but phosph orylates Kemptide and protamine sulfate poorly. Translocation of PKC-z eta from the cytosolic to the particulate membrane fraction upon expos ure of monocytes to LPS provided further evidence for activation of th e kinase. Preincubation of monocytes with the phosphatidylinositol 3-k inase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS- induced activation of PKC-zeta. Furthermore, activation of PKC-zeta fa iled to occur in U937 cells transfected with a dominant negative mutan t Of the p85 subunit of PI 3-kinase, PKC-zeta activity was also observ ed to be enhanced in vitro by the addition of phosphatidylinositol 3,4 ,5P(3). These findings are consistent with a model in which PKC-zeta i s activated downstream of PI 3-kinase in monocytes in response to LPS.